Project Summary The diaphragm is a critical skeletal muscle that separates the thoracic from the abdominal cavity and drives the inspiration phase respiration. Defects in diaphragm development cause congenital diaphragmatic hernias (CDH), a common structural birth defect (1 in 3,000 births) where abdominal contents herniate into the thorax, obstructing lung development and leading to 50% neonatal mortality. Despite the functional significance of the diaphragm and the prevalence of CDH, the biophysical properties underlying hernia formation and the contribution of extracellular matrix (ECM) to the structural integrity of the diaphragm is largely unexplored. This proposal will test the hypothesis that defects in ECM organization lead to alterations in connective tissue stiffness associated with hernias (Aim 1), determine whether connective tissue fibroblasts are a critical source of ECM (Aim 2), and identify ECM components regulated by the transcription factor GATA4, a gene strongly associated with CDH (Aim 3). My recent work has demonstrated how mutations in key regulators of muscle progenitor migration lead to a diaphragm with partial muscle, where regions of connective tissue lack muscle but critically do not herniate. By directly comparing herniated connective tissue (using a previously established model of CDH) to amuscular regions that maintain their structural integrity, I will investigate biomechanical properties as well as ECM composition and organization associated with tensile strength in the diaphragm. This research will identify the embryonic source of key ECM components (K99), clarify how collagen and elastin crosslinking impacts tissue mechanics in the diaphragm (K99, R00) and characterize therapeutic candidates affecting stiffness of diaphragm connective tissue fibroblasts (R00). The proposed experiments will provide me with valuable training in mouse genetics, atomic force microscopy, and mass spectrometry. Under the mentorship of Dr. Gabrielle Kardon, I will gain the experience and training necessary to transition to an independent academic position. To further my career development, I will present my research at conferences, mentor students, attend relevant courses, and publish my findings. My assembled K99 mentorship committee, composed of Drs. Jeff Weiss, Vladimir Hlady, Kirk Hansen, Benoit Bruneau and Kristen Kwan, will provide the necessary expertise to perform biophysical measurements in cells and ex vivo tissues, incorporate these measurements into finite element models of the diaphragm, characterize the ECM profile of diaphragm connective tissue with quantitative precision, and analyze the GATA4 transcriptional network in the developing diaphragm. The Pathway to Independence Award will enable me to pursue an ambitious research program investigating ECM regulation of connective tissue structural integrity in musculoskeletal development.